The mononuclear phagocyte system in Graft-versus-host-disease

Abstract

The human mononuclear phagocyte system of monocytes, macrophages and dendritic cells participates in both innate and adaptive immune responses. However, the accurate identities, functions and inter-relationships of these crucial immune cells during inflammation are poorly defined. Two inflammatory settings were examined in this work: the skin in acute Graft-versus-Host Disease and the lung during experimental inflammation induced by LPS inhalation. The purpose of this enquiry was to characterize inflammatory mononuclear phagocytes in tissue, investigate their origins and explore their contribution to disease pathogenesis. In the skin GvHD study, shave biopsies were obtained from 73 individuals on presentation with acute rash following bone marrow transplantation (BMT). Controls were obtained from 19 BMT recipients at matched time points without rash and 26 healthy individuals undergoing plastic surgery. Tissue was digested and the leukocyte composition analysed by flow cytometry/ sorting. Sorted populations were used in functional assays or in gene expression experiments performed using NanoString technology. An in vitro equivalent of CD14-expressing mononuclear phagocytes (MPs) was developed using HLA-matched mixed leukocyte reactions. Gene expression was measured and the function of these equivalents in a skin explant model of GvHD was tested. GvHD lesional skin was characterized by expansion of CD14-expressing MPs (GVH14) and a reduction in CD1c-expressing MPs. GVH14 were identified as donor monocyte-derived macrophages. Functionally, GVH14 could produce chemokines to recruit T lymphocytes to lesions. They were capable of activating and expanding T lymphocytes in vitro. GVH14 equivalents could damage basal keratinocytes of the epidermis without the presence of T cells. This characterization has identified a novel pathogeneic role for macrophages in acute GvHD. In the LPS inhalation study, 13 healthy individuals received saline (0.9% sodium chloride) and 13 received LPS (0.9% sodium chloride with 2mg LPS from E.coli 026:B6) by dosimeter nebulizer. Blood samples were obtained at 2, 4, 6 and 24 hours following inhalation. Between 7 and 8 hours post-inhalation, bronchoalveolar ! V! lavage (BAL) of a sub-segment of the right middle lobe was performed. BAL fluid supernatant chemokines and cytokines were analysed by multiplexed ELISA. The cellular component of BAL was analysed by flow cytometry/ sorting. Sorted populations were used in functional assays or in gene expression experiments performed using NanoString technology. Seven distinct MPs were identified in steady state (i.e. following saline inhalation). Following LPS inhalation, neutrophils, CD14-expressing MPs and CD1c-expressing MPs were expanded. Phenotypically, CD1c expressing MPs resembled blood cDC2. Both subsets of blood cDC2 were recruited to the airspace but their distinct functions and gene expression profiles converged upon recruitment. This analysis provides the first detailed characterization of BAL fluid MPs. As such it provides a foundation for studying MPs in human lung diseases, including the Idiopathic Pneumonia Syndrome occurring after BMT. It detailed the surprising observation that blood cDC2 can be recruited to tissue in inflammation, challenging the dogma that inflammatory MPs must be monocyte-derived.